1. Field of the Invention
This invention relates to the development of an expression system which is useful in industrial Bacilli to produce target proteins which include, but are not limited to, alkaline proteases, amylases, cellulases, lipases or other hydrolyases which are normally excreted outside of the host cell.
2. Description of the Prior Art
The DNA sequence of a Bacillus licheniformis ATCC 53926 gene encoding a subtilisin Carlsberg type protease has previously been disclosed in European Patent EP0348814 (Jan. 3, 1990) and Jacobs, M. et al. (1985) Nucleic Acids Research 13:8915-8926. This DNA sequence contains a putative stem-loop structure positioned just upstream from the ribosomal binding site (RBS) and the translational initiation codon (FIG. 1). The .DELTA.G of formation of this structure has been calculated to be -16.0 kcal/mole, using the method from Zuker and Stiegler. There was evidence that deletion of a StuI/DraI fragment from the B. licheniformis Carlsberg protease gene, which includes an upstream region and most of the stem-loop, affects protease production by altering, the efficiency of mRNA translation (Jacob, M.,(1987) Poster T-4, Fourth International Conference on Genetics and Biotechnology of Bacilli, San Diego, Calif.). When a premoter inducible by xylose(P.sub.xyl) was cloned into the StuI site in front of the stem-loop, protease mRNA synthesis was detected during exponential growth but protease activity was only detected in the stationary phase of growth (Jacob, M.,(1987). This is he same result as for the native Carlsberg promoter(P.sub.carl). When the StuI/DraI fragment was deleted and replaced by a fragment carrying P.sub.xyl, then Carlsberg protease activity was detected during exponential growth. Similar amounts of full length Carlberg mRNA were detected in the exponential stage of growth for constructs with and without the putative stem-loop structure (Jacob, 1987; data not shown) leading to the conclusion that the effect on protease yield was at the level of translation.